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PubMed2002

Accelerated degradation of mislocalized UDP-glucuronosyltransferase family 1 (UGT1) proteins in Gunn rat hepatocytes.

Gunn rat is a hyperbilirubinemic rat strain that is inherently deficient in the activity of UDP-glucuronosyltransferase form 1A1 (UGT1A1). A premature termination codon is predicted to produce truncated UGT1 proteins that lack the COOH-terminal 116 amino acids in Gunn rat. Pulse-chase experiments using primary cell cultures showed that the truncated UGT1A1 protein in Gunn rat hepatocytes was synthesized similarly to wild-type UGT1A1 protein in normal Wistar rat hepatocytes. However, the truncated UGT1A1 protein was degraded rapidly with a half-life of about 50 min, whereas the wild-type UGT1A1 protein had a much longer half-life of about 10 h. The rapid degradation of truncated UGT1A1 protein was inhibited partially but not completely by treating Gunn rat hepatocytes with proteasome inhibitors such as carbobenzoxy-Leu-Leu-leucinal and lactacystin. By contrast, neither the lysosomal cysteine protease inhibitor nor the calpain inhibitor slowed the degradation. Our findings show that the absence of UGT1 protein from Gunn rat hepatocytes is due to rapid degradation of the truncated UGT1 protein by the proteasome and elucidate the molecular basis underlying the deficiency in bilirubin glucuronidation.

Acetylcysteine↗

PubMed1993

Characterization of the genomic sequence of type V (or 3a) hepatitis C virus isolates and PCR primers for specific detection.

We have identified four new hepatitis C virus (HCV) isolates whose genomic RNA could be amplified by PCR using primers from the 5' untranslated region (UTR), but the RNA could not be detected with genotype I to IV (or types 1a, 1b, 2a and 2b respectively)-specific core region-derived primers. We compared the nucleotide sequences of the new isolates from positions 65 to 1850 (3' end of 5' UTR, C, E1 and 5' end of E2/NS1) and 8276 to 9394 (3' end of NS5 and 3' UTR) with those for genotypes I to IV. The four isolates had the following characteristics: (i) the overall nucleotide sequence similarity between the four isolates was 95 to 96%, compared to 73 to 74%, 73%, 70% or 69 to 70% against genotypes I, II, III or IV, respectively; (ii) the sequence similarity to other reported 'type V (3a)' isolates was 88 to 100%; (iii) the hypervariable region 1 [(HVR)-1] was present but HVR-2 was absent within the E2/NS1 region; (iv) only one in-frame termination codon was present for the presumed polyprotein; (v) the 3'UTR preceding a terminal poly(U) stretch was significantly shorter than in genotype I to IV isolates. We classified the four isolates as genotype V (3a), and searched for uniquely conserved nucleotide sequences that could be used for type-specific PCR. A core region-derived primer pair (no. 104V: 5' CGTAAAACTTCT GAACGGTC, sense and no. 339: 5' GCTGAGCCCA GGACCGGTCT, antisense) was identified and successfully used to diagnose genotype V (3a) HCV infection.

Amino Acid Sequence↗

PubMed1978

Nucleotide sequence of the simian virus 40 Hind-K restriction fragment.

The restriction fragment Hind-K represents 4.2% of the genome of Simian virus 40 (SV40) and is located near the middle of the late region. Its nucleotide sequence is reported here. It was mainly established by analysis of transcription products, synthesized by means of Escherichia coli RNA polymerase and nucleoside triphosphates, one of which was (alpha-32P)-labeled. Strand assignment was possible by hybridization of asymmetric, labeled transcripts of total SV40 DNA to filter-bound Hind-K fragment. Further information and unambiguous confirmation of the sequence was obtained by the use of direct DNA-sequencing methods. For this purpose the fragment was labeled at the 5' ends by means of polynucleotide kinase and [gamma-32P]ATP and redigested with a suitable restriction enzyme. The separated products were then either partially digested with snake venom diesterase for analysis by the 'wandering spot' method or partially degraded with the base-specific reagents dimethylsulphate or hydrazine for direct sequence analysis on gel. The Hind-K sequence is 219 base pairs long. The message strand is particularly rich in adenosine (39%) and purines. The nucleotide sequence cna unambiguously be translated into an amino acid sequence and the N-terminal codon of the viral protein VP1 gene could be identified. The amino-terminal part of VP1 is rich in proline and lysine. The nucleotide sequence of Hind-K codes also for the carboxyl-terminal part of the viral protein VP2 and VP3 genes, which partly overlap the VP1 gene.

Base Sequence↗

PubMedDate not supplied

Molecular characterization of a sarcoplasmic-endoplasmic reticulum Ca+2 ATPase gene from Trichomonas vaginalis.

DNA fragments homologous to P-type cation translocating ATPase genes were identified in Trichomonas vaginalis by polymerase chain reaction (PCR) amplification. The genomic locus corresponding to one PCR fragment, TVCA1, contains a 3,055 base-pair open reading frame encoding a 108,162 dalton protein composed of 981 amino acids. TVCA1 lacks introns, is present in a single copy, and is expressed as a 3.1 kb transcript with short 5' and 3' untranslated regions. Separate primer extension experiments map the 5' end of the TVCA1 transcript to 12 and 16 nucleotide bases (nt) upstream of the methionine initiation codon. The message polyadenylation site is located 62 nt downstream of the protein termination codon at a CA dinucleotide. The TVCA1 protein sequence shares 57-58% similarity with rabbit, schistosome, trypanosome and malarial sarcoplasmic-endoplasmic reticulum calcium (SERCA) pumps, and significantly lower similarity with plasma membrane calcium pumps and cation translocating ATPases of other ion specificities. Structural and functional domains identified in P-type ATPases as well as 61/68 residues specifically implicated in SERCA pump activity are conserved in TVCA1. However, TVCA1 lacks binding sites for phospholamban regulation, thapsigargin inhibition and the calmodulin dependent protein kinase site phosphorylation present in other SERCA pumps.

Amino Acid Sequence↗

PubMed2005

Genetic analysis of Shwachman-Diamond syndrome: phenotypic heterogeneity in patients carrying identical SBDS mutations.

Shwachman-Diamond syndrome (SDS) is a rare hereditary disorder characterized by pancreatic exocrine insufficiency, bone marrow dysfunction and skeletal changes. Recently, the cause of SDS was identified as mutations of Shwachman-Bodian-Diamond syndrome gene (SBDS) and most mutations are caused by gene conversion between SBDS and its highly homologous pseudogene. Clinical variations especially in skeletal and bone marrow abnormalities are well known in this syndrome. To study the relationship between SBDS mutation and its clinical features, we analyzed 9 Japanese patients including one sibling and detected the three different SBDS mutations in 7 patients: a mutation that disrupts the donor splice site of intron 2, deletes 8 bp of the exon 2 and produces premature termination (258+2 T > C), a dinucleotide change that replaces a lysine at 62 nd amino acid to a termination codon (183-184 TA > CT), and a 4-bp deletion that causes premature termination by frameshift (292-295 delAAAG). The 5 patients represent compound heterozygotes of the 258+2 T > C and 183-184 TA > CT mutations. One patient is a compound heterozygote of the 258+2 T > C and 292-295 delAAAG mutations, and in the remaining one case only a 258+2 T > C mutation could be detected. Thus, the 258+2 T > C and 183-184 TA > CT mutations are prevalent among Japanese patients. No mutations were found in two cases, despite the clinical features. Of the 7 patients with SBDS mutations, persistent hematologic abnormalities and skeletal changes were not observed in 3 and 2 patients, respectively. Notably, clinical variations are present even among the patients with the identical genotype: compound heterozygotes of the 258+2 T > C and 183-184 TA > CT mutations. Further study will be required to explain the clinical heterogeneity.

Base Sequence↗

PubMed1992

Nucleotide sequence of the Escherichia coli rfe gene involved in the synthesis of enterobacterial common antigen. Molecular cloning of the rfe-rff gene cluster.

The genetic determinants of enterobacterial common antigen (ECA) include the rfe and rff genes located between ilv and cya near min 85 on the Escherichia coli chromosome. The rfe-rff gene cluster of E. coli K-12 was cloned in the cosmid pHC79. The cosmid clone complemented mutants defective in the synthesis of ECA due to lesions in the rfe, rffE, rffD, rffA, rffC, rffT, and rffM genes. Restriction endonuclease mapping combined with complementation studies of the original cosmid clone and six subclones revealed the order of genes in this region to be rfe-rffD/rffE-rffA/rffC-rffT-rffM . The rfe gene was localized to a 2.54-kilobase ClaI fragment of DNA, and the complete nucleotide sequence of this fragment was determined. The nucleotide sequencing data revealed two open reading frames, ORF-1 and ORF-2, located on the same strand of DNA. The putative initiation codon of ORF-1 was found to be 570 nucleotides downstream from the termination codon of rho. ORF-1 and ORF-2 specify putative proteins of 257 and 348 amino acids with calculated Mr values of 29,010 and 39,771, respectively. ORF-1 was identified as the rfe gene since ORF-1 alone was able to complement defects in the synthesis of ECA and 08-side chain synthesis in rfe mutants of E. coli. Data are also presented which suggest the possibility that the rfe gene is the structural gene for the tunicamycin sensitive UDP-GlcNAc:undecaprenylphosphate GlcNAc-1-phosphate transferase that catalyzes the synthesis of GlcNAc-pyrophosphorylundecaprenol (lipid I), the first lipid-linked intermediate involved in ECA synthesis.

Amino Acid Sequence↗

PubMed1989

Translational readthrough of the murine leukemia virus gag gene amber codon does not require virus-induced alteration of tRNA.

An in vitro system to assay translational readthrough of the UAG termination codon at the murine leukemia virus (MuLV) gag-pol junction was developed by using rabbit reticulocyte lysates programmed by SP6-generated Moloney MuLV gag-pol mRNA. Under conditions in which the suppressor activity of the lysate was dependent on addition of tRNA, it could be shown that readthrough synthesis was stimulated to approximately the same extent by equivalent amounts of tRNA from MuLV-infected and uninfected NIH 3T3 cells. Analysis of glutamine tRNA, which mediates suppression in vivo, showed that the level of glutamine acceptor activity and the chromatographic profile of glutamine isoacceptors were unchanged following virus infection. On the basis of these results, we conclude that the suppressor tRNA occurs normally within the tRNA population of uninfected cells and need not be induced in response to virus infection.

Animals↗

PubMed1994

Three Tetrahymena tRNA(Gln) isoacceptors as tools for studying unorthodox codon recognition and codon context effects during protein synthesis in vitro.

Three glutamine tRNA isoacceptors are known in Tetrahymena thermophila. One of these has the anticodon UmUG which reads the two normal glutamine codons CAA and CAG, whereas the two others with CUA and UmUA anticodons recognize UAG and UAA, respectively, which serve as termination codons in other organisms. We have employed these tRNA(Gln)-isoacceptors as tools for studying unconventional base interactions in a mRNA- and tRNA-dependent wheat germ extract. We demonstrate here (i) that tRNA(Gln)UmUG suppresses the UAA as well as the UAG stop codon, involving a single G:U wobble pair at the third anticodon position and two simultaneous wobble base pairings at the first and third position, respectively, and (ii) that tRNA(Gln)CUA, in addition to its cognate codon UAG, reads the UAA stop codon which necessitates a C:A mispairing in the first anticodon position. These unorthodox base interactions take place in a codon context which favours readthrough in tobacco mosaic virus (TMV) or tobacco rattle virus (TRV) RNA, but are not observed in a context that terminates zein and globin protein synthesis. Furthermore, our data reveal that wobble or mispairing in the middle position of anticodon-codon interactions is precluded in either context. The suppressor activities of tRNAs(Gln) are compared with those of other known naturally occurring suppressor tRNAs, i.e., tRNA(Tyr)G psi A and tRNA(Trp)CmCA. Our results indicate that a 'leaky' context is neither restricted to a single stop codon nor to a distinct tRNA species.

Amino Acyl-tRNA Synthetases↗

PubMed1984

Cloning and sequence analysis of cDNA for rat spleen thymosin beta 4.

Molecular cloning of a cDNA has established the sequence of the translated portion of the mRNA for rat spleen thymosin beta 4. The presence of a methionyl initiator codon immediately preceding the codon for the first seryl residue of mature thymosin beta 4 is consistent with previous results indicating the absence of a signal peptide in the product translated in vitro from rat spleen mRNA. The cDNA sequence analysis also established the presence of two terminator codons immediately following the codon for the COOH-terminal seryl residue. Thymosin beta 4 is thus synthesized as a 5100-dalton peptide containing 44 amino acid residues. Removal of the initiator methionyl residue and acetylation of the NH2-terminal serine residue would yield mature thymosin beta 4 containing 43 amino acids. The absence of a signal peptide makes it unlikely that thymosin beta 4 is a secreted peptide.

Amino Acid Sequence↗

PubMed1999

Missense and nonsense mutations in codon 659 of MLH1 cause aberrant splicing of messenger RNA in HNPCC kindreds.

Germline mutations that give rise to premature termination codons in mRNAs have frequently been associated with aberrant processing of the nascent transcripts. This can take the form either of nonsense-mediated mRNA decay or of aberrant splicing of the pre-mRNA. In a family affected by hereditary nonpolyposis colorectal cancer, a two-nucleotide deletion in codon 659, which introduces a frameshift and a new stop codon in exon 17 of the DNA mismatch repair gene MLH1, has been reported to lead to skipping of the exon. We now report that this phenomenon occurs also when there are missense or nonsense mutations in this codon. Our results thus suggest that in aberrant splicing the nature of the mutation may be less important than its position within the exon. These findings are of importance to mutation interpretation, as they imply that aberrant splicing could be associated even with silent mutations that do not lead to amino acid substitutions. Genes Chromosomes Cancer 26:372-375, 1999.

Adaptor Proteins, Signal Transducing↗

PubMed1987

Molecular cloning of the cDNA for rat spleen thymosin beta 10 and the deduced amino acid sequence.

A rat spleen cDNA library was prepared and employed for the molecular cloning of the cDNA for thymosin beta 10, a peptide that previously had been found to accompany the closely related peptide, thymosin beta 4, in several species of mammals (S. Erickson-Viitanen, S. Ruggieri, P. Natalini, and B. L. Horecker (1983) Arch. Biochem. Biophys. 225, 407-413). First-round screening with a synthetic oligodeoxynucleotide probe yielded 55 positive clones, and sequence analysis of 11 of these clones revealed that they all coded for a peptide containing the thymosin beta 10 sequence, except for an additional arginyl residue at position 39. This peptide, designated thymosin beta 10arg, had been identified previously in rabbit tissues and reported as a variant of thymosin beta 10 (S. Ruggieri, S. Erickson-Viitanen, and B.L. Horecker (1983) Arch. Biochem. Biophys. 226, 388-392). Analysis of the 55 positive clones using a specific oligodeoxynucleotide probe constructed to correspond to the mRNA sequence, including the codon for Arg39, confirmed that they all coded for the amino acid sequence including Arg39. Based on these results, the existence of a molecular species lacking Arg39 is considered unlikely, and we conclude that thymosin beta 10 contains 43, rather than 42, amino acid residues, with identity to thymosin beta 4 in 32 of the 43 residues. We propose that the name thymosin beta 10 be used to refer to the peptide containing Arg39 and that the designation thymosin beta 10arg be dropped. In the cDNA sequence the codons for Ala1 and Ser43 of thymosin beta 10 are flanked by initiator and terminator codons, respectively; thus, both the thymosin beta 4 and thymosin beta 10, which coexist in mammalian cells and tissues, are synthesized without the formation of larger polypeptide precursors.

Amino Acid Sequence↗

PubMed1992

Nucleotide sequence of the 3'-terminal region of potato virus A RNA.

The sequence of the 3'-terminal region of the genome of the potato virus A (PVA) was obtained from two independent cDNA clones. This sequence is 1383 nucleotides long and contains an open reading frame of 1178 nucleotides, ending with the translation termination codon TAA and followed by untranslated region of 205 nucleotides. Since the N-terminal amino acid of the coat protein of PVA was blocked, the position of the putative coat protein cleavage site has been deduced by searching for consensus sequences and by the analogy to other potyviruses. The resulting coat protein is 269 amino acids long and has a calculated MW of 30257. Two independently sequenced cDNA clones show sequence heterogeneity at four nucleotide positions: C422/A422, G432/A432, G446/A446 and T706/C706. Three first nucleotide differences are located at the PVA coat protein N-terminal region and led to the change of the amino acid. The coat protein of PVA displayed significant (73-78%) sequence homology to the coat proteins of six other potyviruses: papaya ringspot virus (PRV), pepper mottle virus (PeMV), plum pox virus (PPV), potato virus Y (PVY), sugarcane mosaic virus (SCMV) and tobacco vein mottling virus (TVMV). Even higher sequence homology (82%) was detected with a coat protein of a seventh potyvirus, tobacco etch virus (TEV). Major differences among the coat protein of PVA and of other potyviruses are located at the N-terminal region of the coat protein.

Amino Acid Sequence↗

PubMed2005

Transcriptional silencing of nonsense codon-containing immunoglobulin minigenes.

Cells possess mechanisms to prevent synthesis of potentially deleterious truncated proteins caused by premature translation-termination codons (PTCs). Here, we show that PTCs can induce silencing of transcription of its cognate gene. We demonstrate for immunoglobulin (Ig)-mu minigenes expressed in HeLa cells that this transcriptional silencing is PTC specific and reversible by treatment of the cells with histone deacetylase inhibitors. Furthermore, PTC-containing Ig-mu minigenes are significantly more associated with K9-methylated histone H3 and less associated with acetylated H3 than the PTC-free Ig-mu minigene. This nonsense-mediated transcriptional gene silencing (NMTGS) is also observed with an Ig-gamma minigene, but not with several classic NMD reporter genes, suggesting that NMTGS might be specific for Ig genes. NMTGS represents a nonsense surveillance mechanism by which truncation of a gene's open reading frame (ORF) induces transcriptional silencing through chromatin remodeling. Remarkably, NMTGS is inhibited by overexpression of the putative siRNase 3'hExo, suggesting that siRNA-like molecules are involved in NMTGS.

Base Sequence↗

PubMed1987

Sequence analysis of the Escherichia coli dnaE gene.

We have determined the sequence of a 4,350-nucleotide region of the Escherichia coli chromosome that contains dnaE, the structural gene for the alpha subunit of DNA polymerase III holoenzyme. The dnaE gene appeared to be part of an operon containing at least three other genes: 5'-lpxB-ORF23-dnaE-ORF37-3' (ORF, open reading frame). The lpxB gene encodes lipid A disaccharide synthase, an enzyme essential for cell growth and division (M. Nishijima, C.E. Bulawa, and C.R.H. Raetz, J. Bacteriol. 145:113-121, 1981). The termination codons of lpxB and ORF23 overlapped the initiation codons of ORF23 and dnaE, respectively, suggesting translational coupling. No rho-independent transcription termination sequences were observed. A potential internal transcriptional promoter was found preceding dnaE. Deletion of the -35 region of this promoter abolished dnaE expression in plasmids lacking additional upstream sequences. From the deduced amino acid sequence, alpha had a molecular weight of 129,920 and an isoelectric point of 4.93 for the denatured protein. ORF23 encoded a more basic protein (pI 7.11) with a molecular weight of 23,228. In the accompanying paper (D.N. Crowell, W.S. Reznikoff, and C.R.H. Raetz, J. Bacteriol. 169:5727-5734, 1987), the sequence of the upstream region that contains lpxA and lpxB is reported.

Amino Acid Sequence↗

PubMed2001

Genome Structure and the p10 Gene of the Helicoverpa armigera Nucleopolyhedrovirus.

The genomic DNA of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (C1 clone) was digested with BamHI, EcoRV, HindIII, KpnI, PstI and XbaI, respectively, and formed 11, 31, 13, 6, 7, and 25 fragments larger than 400 bp, respectively. The size of genome was estimated to be about 130.7 kb. A detailed physical map was constructed for the six restriction enzymes. The five homologous region, hr1, hr2, hr3, hr4 and hr5, and the ten genes including polyhedrin gene (ph), immediate-early gene(ie1), p74, p10, chitinase gene, DNA directed DNA polymerase gene (DNApol), helicase gene, superoxide dismutase gene (sod), alkaline-exonuclease gene (alk-exo), ecdysteroid UDP-glucosyltransferase gene (egt) were identified and their locations in the genome were determined. The genome organization of HaSNPV is quite different from those of other NPVs ever determined. The p10 gene was located in the fragment BamHI-I(1.89 kb) with the transcriptional direction opposite to the polyhedrin gene. Upstream and downstream of the p10 gene were p26 and p74 gene, respectively. The transcriptional direction of p26 is the same as that of p10 gene, and opposite to that of the p74 gene. The ORF encoding p10 was 261 nucleotide long and encoding a putative 87 amino acid polypeptide of 9.3 kD. The immediate upsteam region of the p10 was an A-rich region, and aconserved TAAG motif, associated with transcriptional start sites in other p10 genes, was identified at a site 52 nucleotides upstream of the start codon ATG. A putative polyadenylation signal, AATAAA, was found 20 nucleotides downstream of the termination codon.

Journal Article↗

PubMed1992

A truncated dystrophin lacking the C-terminal domains is localized at the muscle membrane.

A Duchenne muscular dystrophy patient who displayed near-normal dystrophin staining at the sarcolemma with N-terminal, but not with C-terminal, anti-dystrophin monoclonal antibodies was found to have a frameshift deletion of exons 42 and 43. This deletion introduces an early termination codon, and a 225-kD protein was detected by western blotting with N-terminal antibodies only. The results suggest that an N-terminal truncated dystrophin fragment encoded by exon 1-41 is able to associate with the muscle cell membrane. The current idea that the C-terminal domains of dystrophin are important or essential for its integration with the sarcolemma may have to be reexamined in the light of these observations.

Amino Acid Sequence↗

PubMed1990

The molecular genetic analysis of hemophilia A: a directed search strategy for the detection of point mutations in the human factor VIII gene.

A directed-search strategy for point mutations in the factor VIII gene causing hemophilia A was used to screen eight potentially hypermutable CpG dinucleotides occurring at sites deemed to be of functional importance. Polymerase chain reaction-amplified DNA samples from 793 unrelated individuals with hemophilia A were screened by discriminant oligonucleotide hybridization. Point mutations were identified in 16 patients that were consistent with a model of 5-methylcytosine (5mC) deamination. Four new examples of recurrent mutation were demonstrated at the following codons: 336 (CGA----TGA), 372 (CGC----TGC), 372 (CGC----CAC), and 1689 (CGC----TGC). These are functionally important cleavage sites for either activated protein C or thrombin. Further novel C----T transitions were identified in the remaining arginine codons screened (-5, 427, 583, 795, and 1696), resulting in the creation of TGA termination codons. Differences in mutation frequency were found both within and between the CpG sites and between ethnic groups. These differences are assumed to be due to differences in the level of cytosine methylation at these sites, although direct evidence for this inference is lacking.

Base Sequence↗

PubMedDate not supplied

Conservation of an open-reading frame as an element affecting 5' splice site selection.

Splice site selection is a key element of pre-mRNA splicing and involves specific recognition of consensus sequences at the 5(') and 3(') splice sites. Evidently, the compliance of a given sequence with the consensus 5(') splice site sequence is not sufficient to define it as a functional 5(') splice site, because not all sequences that conform with the consensus are used for splicing. We have previously hypothesized that the necessity to avoid the inclusion of premature termination codons within mature mRNAs may serve as a criterion that differentiates normal 5(') splice sites from unused (latent) ones. We further provided experimental support to this idea, by analyzing the splicing of pre-mRNAs in which in-frame stop codons upstream of a latent 5(') splice site were mutated, and showing that splicing using the latent site is indeed activated by such mutations. Here we evaluate this hypothesis by a computerized survey for latent 5(') splice sites in 446 protein-coding human genes. This data set contains 2311 introns, in which we found 10490 latent 5(') splice sites. The utilization of 10045 (95.8%) of these sites for splicing would have led to the inclusion of an in-frame stop codon within the resultant mRNA. The validity of this finding is confirmed here by statistical analyses. This finding, together with our previous experimental results, invokes a nuclear scanning mechanism, as part of the splicing machine, which identifies in-frame stop codons within the pre-mRNA and prevents splicing that could lead to the formation of a prematurely terminated protein.

Algorithms↗